[Show abstract][Hide abstract] ABSTRACT: Non-healing foot ulcers are the most common cause of non-traumatic amputation and hospitalization amongst diabetics in the developed world. Impaired wound neovascularization perpetuates a cycle of dysfunctional tissue repair and regeneration. Evidence implicates defective mobilization of marrow-derived progenitor cells (PCs) as a fundamental cause of impaired diabetic neovascularization. Currently, there are no FDA-approved therapies to address this defect. Here we report an endogenous PC strategy to improve diabetic wound neovascularization and closure through a combination therapy of AMD3100, which mobilizes marrow-derived PCs by competitively binding to the cell surface CXCR4 receptor, and PDGF-BB, which is a protein known to enhance cell growth, progenitor cell migration and angiogenesis.
Wounded mice were assigned to 1 of 5 experimental arms (n = 8/arm): saline treated wild-type, saline treated diabetic, AMD3100 treated diabetic, PDGF-BB treated diabetic, and AMD3100/PDGF-BB treated diabetic. Circulating PC number and wound vascularity were analyzed for each group (n = 8/group). Cellular function was assessed in the presence of AMD3100. Using a validated preclinical model of type II diabetic wound healing, we show that AMD3100 therapy (10 mg/kg; i.p. daily) alone can rescue diabetes-specific defects in PC mobilization, but cannot restore normal wound neovascularization. Through further investigation, we demonstrate an acquired trafficking-defect within AMD3100-treated diabetic PCs that can be rescued by PDGF-BB (2 μg; topical) supplementation within the wound environment. Finally, we determine that combination therapy restores diabetic wound neovascularization and accelerates time to wound closure by 40%.
Combination AMD3100 and PDGF-BB therapy synergistically improves BM PC mobilization and trafficking, resulting in significantly improved diabetic wound closure and neovascularization. The success of this endogenous, cell-based strategy to improve diabetic wound healing using FDA-approved therapies is inherently translatable.
PLoS ONE 01/2014; 9(3):e92667. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although different cranioplasty storage methods are currently in use, no study has prospectively compared these methods. The authors compare freezing and subcutaneous storage methods in a rat model.
Trephine defects (10 mm) were created in 45 Sprague-Dawley rats. The cranial bone grafts were stored in an autologous subcutaneous pocket (n = 15), frozen at -80°C (n = 15), immediately analyzed (n = 12), or immediately replanted into the defect (n = 3). After 10 days of storage, the subcutaneous or frozen grafts were either replanted (subcutaneous, n = 3; frozen, n = 3) or analyzed (subcutaneous, n = 12; frozen, n = 12). Grafts underwent histologic analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase assay, mechanical testing, and micro-computed tomographic imaging.
After 10 days of storage, physiologic assays demonstrated a significant decrease in cellular functionality (e.g., alkaline phosphatase assay concentration: fresh, 18.8 ± 0.77 mM/mg; subcutaneous, 12.2 ± 0.63 mM/mg; frozen, 8.07 ± 1.1 mM/mg; p < 0.012 for all comparisons). Mechanical integrity (maximal load) of fresh grafts was greatest (fresh, 9.26 ± 0.29 N; subcutaneous, 6.27 ± 0.64 N; frozen, 4.65 ± 0.29 N; fresh compared with frozen, p < 0.001; fresh compared with subcutaneous, p = 0.006). Replantation of subcutaneously stored and frozen grafts resulted in limited bony union and considerable resorption after 12 weeks; in contrast, replanted fresh grafts demonstrated bony union and little resorption.
Current preservation methods for interval cranioplasty do not maintain bone graft viability. Subcutaneous storage appears to provide a small advantage compared with freezing.
Plastic and reconstructive surgery 05/2011; 127(5):1855-64. · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: Fat grafting has been used clinically to alleviate the sequalae of unintended radiation to the skin, however the mechanism of this effect is unknown. We hypothesize that it is related to the delivery of adipose derived stem cells and subsequent neovascularization.
Methods: The dorsum of adult wild-type FVB mice was shaved and depilitated. The dorsal skin was then distracted away from the body and radiated (45 Gy) using a Varian 2300 Linear Accelerator. Tissue oximetry and gross photometric analysis were carried out biweekly for the duration of the experiment. 4 weeks following radiation, fat/sham grafts were placed in the dorsal subcutaneous space. Fat grafts consisted of 1.5 cc's of high density lipoaspirate harvested from human donors and processed using the Coleman technique. Sham grafts consisted of 1.5 cc's of sterile saline. Animals were sacrificed at 4 and 8 weeks following fat/sham grafting and their dorsal skin was processed for histologic analysis. Neovascularization was measured by CD31 staining. Fibrosis was assessed using Smad-3 staining, Picrosirius Red staining (Scar-Index), and epidermal thickness measurements.
Results: Chronic ulceration and fibrotic skin thickening stabilized 4 weeks post-irradiation. Alopecia, skin color/texture, and ulceration were improved in fat-grafted mice compared to sham-treated controls when analyzed photometrically. Tissue oximetry demonstrated significantly increased blood oxygenation in treated animals beginning two weeks following grafting (8 weeks post graft 77.11.4% vs 68.52.2%, p<0.03). Vascular density of irradiated skin was increased in fat grafted mice compared to radiated controls at 4 weeks (7.30.04% vs 5.20.09%, p<0.01). Relative intensity of Smad-3 staining was significantly decreased in treated animals at both 4 and 8 weeks (2.770.3% vs 4.98.9%, p<0.01; 3.05.2% vs 5.81.3%, p<0.03). Picrosirius Red staining demonstrated a diminished scar-index in treated animals at both 4 and 8 weeks (.540.05 vs .74.07, p<0.03; .55.06 vs .93.07, p<0.01). Finally, epidermal thickness measurements demonstrated a decreased thickness in treated animals at both 4 and 8 weeks as well (20.61.5m vs 55.25.6 m, p<0.01; 17.61.1m vs 36.36.1 m, p<0.04).
Conclusion: Human fat grafting has a marked phenotypic and experimental impact on radiation skin damage in a murine model. These affects are related to neovascularization and the downregulation of the TGF-β/Smad3 pathway, which results in restoration of normal skin architecture with decreased fibrosis and epidermal thickness.
[Show abstract][Hide abstract] ABSTRACT: Primary alveolar cleft repair has a 41 to 73 percent success rate. Patients with persistent alveolar defects require secondary bone grafting. The authors investigated scaffold-based therapies designed to augment the success of alveolar repair.
Critical-size, 7 x 4 x 3-mm alveolar defects were created surgically in 60 Sprague-Dawley rats. Four scaffold treatment arms were tested: absorbable collagen sponge, absorbable collagen sponge plus recombinant human bone morphogenetic protein-2 (rhBMP-2), hydroxyapatite-tricalcium phosphate, hydroxyapatite-tricalcium phosphate plus rhBMP-2, and no scaffold. New bone formation was assessed radiomorphometrically and histomorphometrically at 4, 8, and 12 weeks.
Radiomorphometrically, untreated animals formed 43 +/- 6 percent, 53 +/- 8 percent, and 48 +/- 3 percent new bone at 4, 8, and 12 weeks, respectively. Animals treated with absorbable collagen sponge formed 50 +/- 6 percent, 79 +/- 9 percent, and 69 +/- 7 percent new bone, respectively. Absorbable collagen sponge plus rhBMP-2-treated animals formed 49 +/- 2 percent, 71 +/- 6 percent, and 66 +/- 7 percent new bone, respectively. Hydroxyapatite-tricalcium phosphate treatment stimulated 69 +/- 12 percent, 86 +/- 3 percent (p < 0.05), and 87 +/- 14 percent new bone, respectively. Histomorphometry demonstrated an increase in bone formation in animals treated with hydroxyapatite-tricalcium phosphate plus rhBMP-2 (p < 0.05; 4 weeks) compared with empty scaffold.
Radiomorphometrically, absorbable collagen sponge and hydroxyapatite-tricalcium phosphate scaffolds induced more bone formation than untreated controls. The rhBMP-2 added a small but significant histomorphometric osteogenic advantage to the hydroxyapatite-tricalcium phosphate scaffold.
Plastic and reconstructive surgery 12/2009; 124(6):1829-39. · 2.74 Impact Factor